EXCEED THESPACE PROVIDED. Amoeboid cell motility, a property of many eukaryotic cells, plays a key role in physiological and pathological processes such as inflammation, wound healing, and metastatic invasion. The purpose of this proposal is to investigate the molecular mechanism of cell crawling using the simple, specialized sperm of the nematode, Ascaris suum, as an experimental system. These cells display the same cycle of leading edge protrusion, substrate attachment, and cell body retraction as conventional crawling cells but lack the actin machinery usually associated with cell migration. Instead, the motility apparatus of sperm is based on major sperm protein (MSP) filaments that assemble along the leading edge and disassemble at the base of the lamellipod. These unique filaments have no structural polarity indicating that molecular motor proteins are not required for sperm motility. The relationship of MSP cytoskeletal dynamics to locomotion suggests that the forces for leading edge protrusion and cell body retraction are mechanically coupled. This hypothesis will be tested by analysis of an in vitro motility system that reconstitutes both protrusion and retraction. The components parts of the motility apparatus will be identifed and characterized by biochemical and molecular methods seeking to define the minimum requirements for movement and to reconstitute motility from purfied proteins, EMtomography and single-molecule fluorescence assays, including fluorescence speckle microscopy, will be used in parallel to examine the organization of the motility apparatus and explore how its constituent MSP filaments rearrange during protrusion and retraction. Information from biochemical, biophysical, and structural studies will be integrated by assessing the effects of addition, depletion, and mutation of accessory components on motility and correlating alterations of movement with changes in the organization and dynamics of the filaments in the motility apparatus. This strategy will enable a stepwise analysis of motility starting at the level of simple protein-protein interactions and building to an understanding how a network of such interactions produces movement. The long range goal of this project is to define the exact molecular mechanism of sperm locomotion so that comparison MSP and actin-based systems can be used to understand the basic principles of amoeboid cell motility.